Gel Electrophoresis

Gel Electrophoresis

• Technique used for separation of– Deoxyribonucleic acid (DNA)– Ribonucleic acid (RNA)– Protein molecules

• Using an electric current applied to a gel matrix

Gel Electrophoresis

• Uses electricity to separate charged molec. on a gel slab– Separation based on size, shape and charge

• Gel:– Powdered agarose (carb. derived from seaweed)– Dissolve in boiling buffer soln. – Most common agarose is polyacrylamide (PAGE)– Gel solidify and placed in get box and covered with

buffer soln.

Gel Electrophoresis

Gel Electrophoresis

Separation

• “Gel” refers to matrix used to contain, then separate target molecules– Gel is a crosslinked polymer whose composition and porosity is

chosen based on specific weight and composition of target to be analyzed

• "Electrophoresis" refers to electromotive force (EMF) that is used to move molecules through gel matrix– By placing molecules in wells in gel and applying an electric

current, molecules will move through matrix at different rates– Usually determined by mass

• Toward the positive anode if negatively charged• Toward the negative cathode if positively charged

Visualization After Electrophoresis Is Complete….

• Molecules in gel can be stained to make them visible– Ethidium bromide– Silver Stain– Coomassie blue dye

• Other methods may also be used to visualize separation of mixture's components on gel– If analyte molecules fluoresce under ultraviolet light, a

photograph can be taken of gel under ultraviolet lighting conditions

Visualization After Electrophoresis Is Complete….

• If several mixtures have initially been injected next to each other, they will run parallel in individual lanes

• Depending on number of different molecules– Each lane shows separation of components

from original mixture as one or more distinct bands

– One band per component

Visualization After Electrophoresis Is Complete….

• Incomplete separation of components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components

• Bands in different lanes that end up at same distance from top contain molecules that passed through gel with same speed– usually means they are approximately the same size

• There are molecular weight size markers available that contain a mixture of molecules of known sizes– Marker run on one lane in gel parallel to unknown samples– Bands observed can be compared to those of unknown in to

determine their size

Visualization After Electrophoresis Is Complete….

Agarose gel prepared for DNA analysis• The first lane contains a DNA ladder for sizing• Other four lanes show variously-sized DNA fragments that are

present in some but not all of the samples

Applications

• Gel electrophoresis is used in:– Forensics– Molecular biology– Genetics– Microbiology– Biochemistry

• Results can be analyzed quantitatively by visualizing gel with UV light and a gel imaging device

Nucleic acids

• Direction of migration– From negative to positive electrodes– Due to naturally-occurring negative charge

carried by their sugar-phosphate backbone

• Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis

Proteins

• Proteins, unlike nucleic acids, can have varying charges and complex shapes– They may not migrate into gel at similar rates, or at all, when

placing a negative to positive EMF on sample

• Proteins are usually denatured in presence of a detergent such as sodium dodecyl sulfate (SDS)– SDS coats proteins with a negative charge– Amount of SDS bound is relative to size of protein (usually 1.4g

SDS per gram of protein), so that resulting denatured proteins have an overall negative charge

• Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Proteins

SDS-PAGE - The indicated proteins are present in different concentrations in the two samples

DNA Electrophoresis

• Analytical technique used to separate DNA fragments by size

• An electric field forces fragments to migrate through a gel

• DNA molecules normally migrate from negative to positive potential due to net negative charge of phosphate backbone of DNA chain

• At scale of the length of DNA molecules– Gel looks much like a random, intricate network– Longer molecules migrate more slowly because they are more

easily 'trapped' in network• After separation is completed

– Fractions of DNA fragments of different length are often visualizing a fluorescent dye specific for DNA

– Ex: ethidium bromide

DNA electrophoresis• Fragment size is usually reported in "nucleotides", "base

pairs" or "kb"– Fragment size determination is typically done by comparison to

commercially available DNA ladders containing linear DNA fragments of known length

• Types of gel most commonly used for DNA electrophoresis are:– Agarose (for relatively long DNA molecules)– Polyacrylamide (for high resolution of short DNA molecules)

• Gels have conventionally been run in a "slab" format• DNA strand is cut into smaller fragments using DNA

endonuclease– Samples of DNA solution (DNA sample and buffer) are placed in

wells of gel and allowed to run for some time– The less the voltage of electophoresis, the longer time for DNA

sample to run through gel, and this results in a more accurate separation

Migration of DNA Fragments in Agarose• Fragments of linear DNA migrate through

agarose gels with a mobility inversely proportional to log10 of their molecular weight– In other words, if you plot distance from well

that DNA fragments have migrated against log10 of either their MW or number of base pairs, a roughly straight line will appear

• Circular DNA migrate in agarose distinctly differently from linear DNAs of same mass– Uncut plasmids will appear to migrate more

rapidly than same plasmid when linearized– Image shows an ethidium-stained gel with

uncut plasmid in left lane and same plasmid linearized at a single site in right lane

Migration of DNA Fragments in Agarose

• Several additional factors have important effects on mobility of DNA fragments in agarose gels:– 1. Agarose Concentration– 2. Voltage– 3. Electrophoresis Buffer– 4. Effects of Ethidium Bromide

1. Agarose Concentration• Using different concentrations gels resolve

different sizes of DNA fragments– Higher concentrations of agarose for small DNAs– Low agarose concentrations allow resolution of

larger DNAs

• Image shows migration of a set of DNA fragments in 3 concentrations of agarose– All in same gel tray and electrophoresed at same

voltage and for identical times– Notice:

• Larger fragments are much better resolved in 0.7% gel

• While small fragments separated best in 1.5% agarose

• 1000 bp fragment is indicated in each lane

2. Voltage

• As voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments– So, for best resolution of fragments larger

than about 2 kb is attained by applying no more than 5 volts per cm to gel

– The cm value is distance between two electrodes, not the length of the gel

3. Electrophoresis Buffer

• Several different buffers have been recommended for electrophoresis of DNA– Most commonly used for DNA are:– TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA)

• DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength

• Buffers not only establish a pH, but provide ions to support conductivity– If you mistakenly use water instead of buffer, there will be

essentially no migration of DNA in the gel– If you use concentrated buffer (e.g. a 10X stock solution),

enough heat may be generated in gel to melt it.

4. Effects of Ethidium Bromide

• Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels– It can be incorporated into agarose gels, or

added to samples of DNA before loading to enable visualization of fragments within gel

– Binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility

Agarose & Polyacrylamide

• Most commonly used support matrices• Provide a means of separating molecules by size

(porous gels)• Porous gel may act as a sieve by retarding, or in some

cases completely obstructing, movement of large macromolecules while allowing smaller molecules to migrate freely– Dilute agarose gels are generally more rigid and easy to handle

than polyacrylamide of same concentration– Agarose is used to separate larger macromolecules such as

nucleic acids, large proteins and protein complexes– Polyacrylamide, which is easy to handle and to make at higher

concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation

Agarose & Polyacrylamide

• Handout

Separation of Proteins and Nucleic Acids

• Proteins are amphoteric compounds– Their net charge therefore is determined by pH of medium in

which they are suspended• In a solution with a pH above its isoelectric point, a protein has a net

negative charge and migrates towards anode in an electrical field• Below its isoelectric point, protein is positively charged and migrates

towards cathode

• Nucleic acids remain negative at any pH used for electrophoresis– Carry a fixed negative charge per unit length of molecule,

provided by the PO43- group of each nucleotide of nucleic acid

– Electrophoretic separation of nucleic acids is strictly according to size

SDS- PAGE OF PROTEINS

• Separation of Proteins under Denaturing conditions– SDS is an anionic detergent which denatures proteins

by "wrapping around" polypeptide backbone– SDS confers a negative charge to polypeptide in

proportion to its length– In denaturing SDS-PAGE separations migration is

determined not by intrinsic electrical charge of polypeptide, but by molecular weight

Determination of Molecular Weight• Done by:

– SDS-PAGE of proteins– PAGE or agarose gel electrophoresis of nucleic acids - of known

molecular weight along with protein or nucleic acid to be characterized

• A linear relationship exists between logarithm of molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf

• Rf is calculated:– As ratio of distance migrated by molecule to that migrated by a

marker dye-front• A simple way of determining relative molecular weight by

electrophoresis (Mr):– Plot a standard curve of distance migrated vs. log10MW for

known samples– Read off logMr of sample after measuring distance migrated on

same gel

Agarose

• Polysaccharide extracted from seaweed– Typically used at concentrations of 0.5 to 2%– The higher agarose concentration the "stiffer" the gel

• Agarose gels are extremely easy to prepare:– You simply mix agarose powder with buffer solution, melt it by

heating, and pour gel. It is also non-toxic

• Agarose gels have a large range of separation, but relatively low resolving power

• By varying concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques

Polyacrylamide

• Cross-linked polymer of acrylamide– length of polymer chains is dictated by the concentration of

acrylamide used– Typically between 3.5 and 20%

• Polyacrylamide gels are more annoying to prepare than agarose gels– Because oxygen inhibits polymerization process, they must be

poured between glass plates (or cylinders)

• Acrylamide is a potent neurotoxin and should be handled with care!– Polyacrylamide is considered to be non-toxic, but

polyacrylamide gels should also be handled with gloves due to possible presence of free acrylamide

Polyacrylamide

• Polyacrylamide gels have a rather small range of separation, but very high resolving power– Case of DNA, polyacrylamide is used for separating

fragments of less than about 500 bp– Under appropriate conditions, fragments of DNA

differing is length by a single base pair are easily resolved

– In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

• Gel stains:– Nucleic acids are colorless– Must be stained– DNA stains:

• Ethidium bromide (EtBr)…orange when mixed with DNA under UV light

• Methylene blue…dark blue…not as sensitive as EtBr viewed with white light

Gel Electrophoresis

Only one DNA type

most common Sizing standard

Plasmid restriction digestionDNA sample from bacterial

chromosome RNA

Smears (thousands of different size molec in small concentration)

No nucleic acids

DNA so large will not load

Ex: eukaryotic genome

Proteins

• Companies that produce protein products or study proteins must be able to:– Separate the protein of interest– Determine that amount of protein present.

• Characteristics of proteins that make it possible to achieve either one of both points above:– Overall charge, size, shape, and solubility

Proteins

• SDS-PAGE– Gel electrophoresis allows for the separation

of proteins based on charge, size, and shape.– Polyacrylamide gel electrophoresis is utilized

(PAGE).• Allows for better resolution• 4-18% gels most commonly used

– Higher concentration for smaller proteins

• When protein size unknown gradient gels can be used.

– Less concentrated at the top than the bottom

Proteins

• SDS-PAGE– Use of sodium dodecyl

sulfate (SDS)• Denatures proteins into

polypeptide strands• Gives each polypeptide

strand an overall negative charge

• Proteins studied are strictly being separated by size

Proteins

• SDS-PAGE– Visualization of proteins in

gel• Coomassie Blue

– Milligram amounts of protein.

• Silver stain– Microgram amounts of

protein.

– Size of unknown bands can be determined from comparison to protein molecular weight standard

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